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INSULINAND GLUCAGONRESPONSESIN THE BLACKBEAR HIBERNATING P.J.PALUMBO,Mayo Clinic, Mayo Foundation, and Mayo Medical School, Rochester, MN55905 DIANNEL. WELLIK, College of Medicine and Carle Foundation Hospital, University of Illinois, Urbana, IL61801 NANCYA. BAGLEY,Mayo Medical School, Rochester, MN55905 RALPHA. NELSON,College of Medicine and Carle Foundation Hospital, University of Illinois, Urbana, IL61801 Abstract: To study the hormonal changes during hibernation in the black bear (Ursus americanus), 2 adult male bears were injected with insulin and with glucagon during their active and hibernating phases, and plasma urea, glucose, insulin, glucagon, and corticosteroidswere measuredbefore and after. The baselineurea values decreasedduringhibernationa patternconsistentwith proteinconservation. Baselineinsulinvalues increasedduringthe fall active phase, when bearsare hyperphagic,returningto the normalrangeduringearly hibernation. Baselineglucagonlevels increasedduringthe fall hyperphagiaphase and early hibernation and then tended to decrease at the end of hibernation. Baseline corticosteroid levels were lower during the summer active phase than during the other three periods. The insulin, glucagon, and corticosteroid responses to glucagon and insulin injection were variable,but in generalweredelayedduringearlyhibernation.The plasmaglucoseresponseto insulinstimulationwas also delayed duringearly and late hibernationbut more so duringearlyhibernation. The glucoseresponseto glucagonstimulationwas delayed to similardegreesduringboth early and late hibernation. These findingsare consistentwith decreasedglucose utilizationand increasedlipolysisduringhibernation. Furthermore,the apparentincreasein glucose utilizationat the end of hibernationwhen fat storesare nearlyexhaustedsuggestsa continuumof metabolicactivityfrom earlyto late hibernationwith a transitionto the active phaseby the end of hibernation. Int. Conf. Bear Res. and Manage. 5:291-296 Metabolic changes are noted during hibernation in the black bear while the bear maintains nearly normal (within 2 C) body temperature (Nelson 1973, Nelson et al. 1975, Folk et al. 1976). Although the bear does not eat, drink, urinate, or defecate during the months of hibernation, it does not become uremic or dehydrated, and emerges from hibernationwith a loss of 15 to 20% of prehibernation body weight. Body composition studies have shown that this weight loss is due almost exclusively to loss of body fat (Nelson et al. 1975, Lundberget al. 1976). The blood glucose level decreases only slightly during the winter compared to glucose concentrationin the summer, but in the active starving bear, blood glucose levels are markedlyreduced (Nelson et al. 1975, Nelson 1978). Current evidence suggests that in small animals, hibernation is controlled by the hypothalamic-pituitaryaxis. Adrenocorticotrophic (ACTH) blood concentrations decrease in winter hibernation in the European hedgehog In the black bear, the examination of hypothalamic-pituitaryfunction is complicatedby lack of specific antisera for bear pituitary hormones such as thyrotrophin (TSH), growth hormone (GH), and ACTH. Triiodothyronine(T3) levels decrease during the winter, but the thyroid gland shows normal histology (Seal et al. 1970, Nelson et al. 1973). Previous studies involving measurements of TSH by bioassay and thyroxine (T4) and T3 indicatedthat the black bear manifests hypothalamic hypothyroidism during hibernation (Azizi et al. 1977). Whether hypothalamichypothyroidism or a generalized hypothalamichypofunction with secondaryhypopituitarismaccounts for the changes in the metabolism of carbohydrate, protein, and fat reportedpreviouslyduring hibernationis not known. Insulin and glucagon stimulation studies were carried out during active and hibernatingphases of the black bear's life cycle in order to further elucidate hypothalamicpituitary function during these periods. Plasmaglucose, insulin, glucagon, (Erinaceus europaeus) (Hoo-Paris 1971), correlat- and corticosteroids were measured during these ing with a decrease in sympathetic neurotrans- tests. Measurementof GH and ACTH was also mitters (Sauerbierand Lemmer 1977). Ground attempted, but lack of cross-reactivitywith the squirrels show a marked reduction in thyroid specific human antiseraused in the assay precludfunction during hibernationcoupled with increas- ed assessment of these hormone levels. ing serotonin levels (Hudson and Deavers 1976, The authors would like to thank the laboratory Hulbert and Hudson 1976). Experimentally,de- of Dr. Vay L.W. Go, at the Mayo Clinic, Rochescreasing serotonin levels will disrupt hibernation ter, Minnesota, and specifically Dan Kluge, for (Spaffordand Pengelley 1971). performing the glucagon assays. Additional 291 292 HORMONERESPONSESIN HIBERNATINGBEARS * Palumbo et al thanks are extended to Dr. Paul Zollman and the staff of the Institute Hills Farm in Rochester, Minnesota, for their invaluableassistance in handling the bears. METHODS Two adult male black bears were studied for 2 consecutive years (1977-1978) at the Institute Hills Farm and ResearchCenter (Mayo Clinic) in Rochester, Minnesota. Bear 1, who weighed 190 kg at the beginning of the studies, had been in captivity at this institute for 8 years, and bear 4, who weighed 145 kg, had been confined for 3.5 years. Summer bear quarterswere double-mesh cages with a boxed-in corner for sleeping and shade. The bears were fed once daily and had free access to water. Winter bear dens were located in a vegetable cold-storagecellar on the east side of a hill. Each bear was placed in a separateculvert with bars on either end and providedwith straw bedding. The bears were not fed or given water while in the winter quarters. Each bear, acting as its own control, was studied during the summer (June) and fall (October) active phases, and duringearly (January)and late (March) hibernationphases. In both active and hibernatingexperiments, blood samples were obtained in bears anesthetized intramuscularlywith promazine and phencyclidine via a syringe and pole assembly. Two baseline blood samples (30 ml/bear) were drawnat 30 minutes and 5 minutes priorto injection of insulin or glucagon and analyzedfor plasma glucose, urea, insulin, glucagon, and corticosteroids. The insulin stimulation experiment was performed by injection of 0.2 units (U) of insulin per kg of body weight into a femoral vein. A unit of insulin representsthe biologicalactivity of 1/22 mg of pure crystalline insulin. Additional blood samples were drawnfrom either the femoral vein or artery, starting on the side opposite the injection site. Samples were obtained at 5, 15, 30, 60, 90, 120, 150, and 180 minutes after administrationof insulin and were analyzed for glucose, insulin, glucagon, and corticosteroids. The glucagonstimulationexperiment was done in the same manner;2 mg of glucagonwas inject- ed intravenously. Serial blood samples were drawnas for the insulin stimulationexperiment. In the 1st year, the insulin stimulation experiment was performed7 days priorto the glucagon stimulation experiment and in the 2nd year, 14 days after the glucagon stimulation experiment. This eliminatedthe possibilitythat the results obtained were related to the sequence in which the tests were done. As a control experiment, insulin levels were also measured following intravenous injection of normal saline (2 ml) in 1 of the bears during the fall active period. Glucose was determined using a Beckmanglucose analyzer. Urea was measured by a standard enzymatic method utilizing urease (Marsh et al. 1957). Levels of insulin, glucagon, and corticosteroids (total of cortisol, corticosterone, and 11deoxycortisol) were measured by previously described methods (Hales and Randle 1963, Murphy 1967, Faloonaand Unger 1974). Statisticalanalysis of the data utilized the standard t-test with significancedeterminedat the 5% level. RESULTS The blood parametersmeasured were similar for the 2 bearsduring both active and hibernating experiments, so data are expressed as the mean of the values for both bears. Baseline values Several differences were seen in the prestimulationblood values during active and hibernationphases (Table 1). During the early hibernation phase, the mean glucose level decreased slightly but significantly (P < 0.005) from the active phase levels. During late hibernation, however, the glucose levels again rose to active phase levels. The decrease in plasma urea during hibernation was quite spectacular, and both early and late hibernationvalues were significantly lower than active values (P < 0.001). The highest mean blood insulin level was seen during the fall active phase, but there were no significantdifferences in insulin levels among any of the experimentalphases. Glucagon levels varied widely, but they tended to be highest during the early hibernationphase; again, no statistically significant differences were noted among any of INHIBERNATING BEARS* Palumbo et al. RESPONSES HORMONE 293 bears prior to insulin and glucagon stimulation experiments during active and hiber- Ta ble1.ackBlood valuesa in 2 confined male nating phases. Hibernating phase Active phase Blood parameter Summer Fall Early Late Glucose (mg/dl) 74-+-5(63-82) 71+?2 (68-76) 60? 1 (59-62) 71?6 (61-87) Urea (mg/dl) 44?5 (34-53) 44?4 (36-55) 8?1 (6-11) 12?1 (9-14) Insulin (,uU/ml) 14?6 (3.0-25) 53? 10 (24-66) 17?3 (8.5-21.5) Glucagon (pg/ml) 78? 13 (57-100) 129?27 (50-164) 175?48 (93-303) 112? 31 (62-161) Corticosteroids (j/g/dl) 1.8?0.2 (1.0-2.0) 7.4? 0.2 (1.3-13) 7.8? 2.5 (2.6-13) 7.6? 1.5 (5.4-12) 32?7 (20-44) a Mean + standarderrorof mean of 4 values (rangein parenthesis). the phases. Corticosteroid levels were similar during fall active and early and late hibernation phases. However, the summer active corticosteroid level was significantly lower (P < 0.02) than the mean value for the other 3 phases. Insulin Stimulation Experiment The plasma insulin response to insulin injection, measured during the fall active and early hibernation phases, revealed a delayed disappearance of exogenous insulin during the hibernation phase. During both phases, maximum plasma insulin levels occurred at 5 minutes after insulin injection. The mean maximum values (N = 2) were 3600%of baseline during the fall active and 12,000%of baseline during the early hibernation phase. During the fall active phase, the insulin levels dropped to baseline values by 90 minutes, while during the early hibernation phase, the mean insulin level (N = 2) was still 800% of baseline at 180 minutes after insulin injection. % of Baseline 100 PA I\ Active Phase * 6/76 o 10/77 Insulin stimulation produced a prompt drop in plasma glucose levels (Fig. 1) with the lowest levels occurring within 30 minutes after insulin injection during the summer and fall active phases, and also during the late hibernationphase. In contrast, during the early hibernation phase, the glucose response was delayed and the maximum decrease was greater. The return to baseline glucose levels also took longer during the early hibernationphase than during the other 3 periods. Plasma glucagon response to insulin stimulation (Fig. 2) during both early and late hibernation phases was delayed from that during the 2 active phases. This is demonstrated by both a later achievement of maximum glucagon values and by a failure to return to baseline levels by 180 minutes during the hibernation phase. The maximum glucagon level achieved during the summer active phase was much larger than the maximum value reached during any of the other 3 phases. % of Baseline 2000 Hibernating Phase * 1/78 o 3/77 80 1600 .0 60 / 400 20 a I Active Phase * 6/76 10/77 \ I 800 nIa * 1/78 o 3/77 1200 40- 30 Hibernating Phase II Ia 120 60 90 150 Time After Injection (min) / aI 180 Fig. 1. Plasma glucose response (mean of 2 values) after insulin injection in 2 confined male black bears during active and hibernating phases. 30 90 120 150 60 Time After Injection (min) 180 Fig. 2. Plasma glucagon response (mean of 2 values) after insulin injection in 2 male black bears during active and hibernating phases. 294 INHIBERNATING BEARS* Palumbo et al. RESPONSES HORMONE Table 2. Plasma corticosteroid levels (mean of 2 values, in jAg/dl)following intravenous injection of insulin (0.2 units/kg of body weight) or glucagon (2 mg/kg) in 2 confined male black bears during active and hibernating phases. Minutesafterinsulin injection 120 90 60 0 Phase Active: Summer Fall 1.5 7.2 24 9.6 24 4.6 Hibernation: Early Late 3.6 5.8 13 18 12 23 Plasma glucagon levels after glucagon injection were measuredduring the fall active and early hibernationphases. The mean maximum glucagon level (N = 2), which occurred 5 minutes after injection, was 1900%of baseline during the fall active phase and 2500% of baseline during the early hibernationphase. Return to baseline glucagon levels during the hibernationphase was delayed, with a mean level of 700%of baseline (N = 2) at 180 minutes after injection comparedto a mean level of 240%of baseline (N = 2) at 180 minutes duringthe active experiment. The degree of corticosteroidresponse to glucagon injection (Table 2) was much lower than the response seen after insulin injection for all 4 1 Hibernating Phase * \ \ 1/78 0 3/77 180 Fig. 3. Plasma glucose response (mean of 2 values) after glucagon injection In 2 male black bears during active and hibernating phases. 12 12 3.5 14 11 22 4.7 7.2 of the experimental phases. Indeed, it appeared that during the early hibernationphase, there was no corticosteroidresponse at all. Plasma glucose response to glucagon administration (Fig. 3) was similar in the summer and fall active phases. The glucose responses seen during the early and late hibernationphases were also similar to each other; however, the maximum glucose levels achieved were higher, and both the rise in glucose levels and the return to baseline values were delayed when compared to the active phase experiments. The insulin levels following glucagon injection (Fig. 4) exhibited a more variableresponse than any of the other parametersmeasured. Although the maximum insulin levels occurredat different times for all of the study periods, they were similar in degree for all of the phases except the late hibernation phase. The insulin response during the late hibernation phase appeared to be suppressed. During the early hibernationphase, the return to baseline values was delayed when comparedto the patternfor the other 3 phases. % of Baseline 16001200- 120 150 90 60 30 Time After Injection (min) 6.2 6.8 12 9.2 14 Glucagon Stimulation Study Minutesafterglucagoninjection 120 90 60 2 7.6 3.3 Plasma corticosteroidresponse to insulin stimulation (Table 2) paralleledthe glucagonresponses, with an increase in corticosteroidlevels at 60 minutes for all study phases. Again, the maximum value achieved was greatest during the summer active phase. %of 2201 Baseline 0 Hibernating Phase * 1/78 - \ 30 3/77 Active Phase * 6/76 oO/77 150 120 90 60 Time After Injection (min) 180 Fig. 4. Plasma insulin response (mean of 2 values) after glucagon injection in 2 male black bears during active and hibernating phases. HORMONE RESPONSES INHIBERNATING BEARS* Palumbo et al. Saline Control Study There was a slight increase in the plasma insulin level 5 minutes after injection of saline in 1 bear during the fall active phase (from 23 ,u U/ ml at time 0 to 40 A U/ml at 5 minutes). Insulin levels returned to the baseline value by 14 minutes and remained there until the end of the 60minute experiment. Such an increase would account for little, if any, of the changes seen during the stimulationtests. DISCUSSION Hibernation in the black bear produced changes in basal and stimulated levels of plasma glucose, insulin, glucagon, and corticosteroids. These alterationsmay be related to hypothalamic hypofunctiontriggeredby external environmental factors. Whether the changes described were influenced by biochemicalor hormonal factors, released in response to external stimuli but not measured, could not be ascertained from these studies. Previous measurements of serum thyroxine and thyrotrophinduring active and hibernating periods indicated hypothalamic hypothyroidism (Azizi et al. 1977). In the present studies, basal urea levels during hibernation were significantly decreased, a finding consistent with previous reports of protein conservation during hibernation (Nelson et al. 1975, Lundberget al. 1976). Basal levels of glucagon and corticosteroids tended to be higher during hibernation, which is consistent with the adaptationof the bear to starvation. This adaptation apparently permits the bear to utilize fat stores rather than protein for energy needs, with a consequent decrease in blood urea. The delay in insulin disappearanceduring hibernationwas consistent with the prolongationof the decrease in plasma glucose observed during hibernation after the intravenous administration of the insulin. These findings suggest that insulin degradationis impairedduring hibernationby mechanism(s) as yet unknown. One possible explanation is that the decrease in the glomerular filtration rate during hibernation (Brown et al. 1971) results in a decrease in insulin clearanceby the kidney. It would be interesting to speculate that the delay in insulin disappearancemay help 295 to promote protein conservation and modulate the release of free fatty acids to be utilized for energy during hibernation. The glucose response to insulin stimulation suggests that in early hibernation, the levels of the antagonistichormones are reduced or the responses of these hormones to stimulation are suppressed, suggesting hypothalamic hypofunction. The rise in plasma glucagon after insulin administrationwas prolongedduring hibernation, perhapsin response to the prolongeddecrease in plasma glucose levels during hibernation. However, the tendency for the plasmaglucagon levels to remain elevated with insulin stimulationis also consistent with fat mobilizationduring this period. The delay in glucagon disappearanceafter glucagon stimulationduring hibernationwas consistent with the findings during insulin stimulation. The rise in plasma glucose with glucagon administration during hibernation was delayed and relatively higher than in the active phase of the bear's life cycle. This difference in plasma glucose response between hibernatingand active phases may be related to the delay in the disappearanceof injected glucagonduring hibernation. The findings suggest a decrease in glucose utilization during hibernation as might be expected when an exogenous fuel or energy source is not available or utilized. This decrease in glucose utilizationis accompaniedby proteinconservation and fat utilizationas the sole source of energy, a most remarkablephenomenon. Ketosis does not develop as a consequence of fat combustion in hibernatingblack bears, presumablyas a result of increasedtriglycerideturnover (Nelson 1980). The rise in plasmainsulin after glucagon injection was most marked in the fall active, hyperphagic phase, prior to hibernation. During early hibernationthis rise in plasma insulin decreased until in later hibernation and summer active phases the patterns of plasma insulin response were quite similar. These findings describe a shifting continuum of metabolic activity from early to late hibernationto the active phase. The corticosteroidlevels after stimulation with insulin and glucagon suggest hyporesponsiveness of the pituitary-adrenalaxis as the bears go into hibernation. This hyporesponsiveness may in turn be a consequence of hypothalamic hypofunction. 296 HORMONE RESPONSES BEARS* Palumbo et al. INHIBERNATING LITERATURE CITED AZIZI,F., J. MANNIX,ANDR.A. NELSON.1977. Thyroid function in the bear before, during and after winter sleep. Clin. Res. 25:289a. D.J. ANDREW,AND U.S. BROWN,D.C., R.O. MULHAUSEN, SEAL. 1971. Renal function in anesthetized dormant and active bears. Am. J. 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